Cells are a field that has developed very rapidly in pharmaceutical research and development in recent years, and its main methods are immune cells and stem cells. Stem cells are divided into embryonic stem cells and adult stem cells, which can be divided into totipotent stem cells, pluripotent stem cells, and unipotent stem cells according to different differentiation potentials.
Induced pluripotent stem cell (iPSC) technology is known as the third medical revolution after drugs and surgery, and is a key development area of the international medical frontier in recent years, providing a new research direction for the entire field of stem cell biology and clinical regenerative medicine.
Definition of IPSC
Induced pluripotent stem cells (iPSCs), also known as artificial induced pluripotent stem cells, are a pluripotent stem cell that introduces a series of inducible factors into mature somatic cells and reprograms them into pluripotent stem cells with characteristics similar to embryonic stem cells.
iPSC can produce syngeneic control cell lines through CRISPR Cas9 gene editing, which are very similar to embryonic stem cells in terms of morphology, gene and protein expression, epigenetic modification status, cell multiplication ability, differentiation ability, etc., and have the same application potential as embryonic stem cells.
Advantages of IPSC:
IPSCTumorigenicity factorMuch lower than embryonic stem cells and mesenchymal stem cells.
iPSCs can be directly induced by adult cellsIt breaks the unavoidable limitations of ethics and immune rejection in stem cell researchIt provides a new perspective for the clinical application and research of stem cells.
iPSCs are the same as other stem cells in the human bodyIt has super differentiation and regeneration ability and low immunogenicity. It has a wide range of applications in the fields of regenerative medicine, screening and development of new drugs.
It is obtained by the patient's own cell induction, thus greatly solving the problem of potential immune rejection.
Application of IPSC
Regenerative medicine: iPSC has a strong ability to differentiate, and under specific conditions, it can induce different types of cells and even organoids required for differentiation
Disease modeling: Due to the inherent self-renewal nature of iPSCs and their potential to differentiate into virtually any cell type in vivo, patient-specific iPSCs can provide a large number of disease-related cells and a variety of different types of cells (such as neurons and cardiomyocytes) that were previously unavailable, enabling personalized disease modeling, which will become a core part of precision medicine;
New drug development: Researchers can use cultured stem cells to test the efficacy of drugs and understand the potential of drugs**, which is unpredictable for researchers to develop new drugs.
For patients with disease and aging, induced pluripotent stem cells are managed by iPSC regenerative medicine, and induced pluripotent stem cells are injected by interventional means to restore the function of cells and organs.
iPSC culture guide
Highlights:
1.Antibiotics are not recommended during iPSC culture and can affect cell viability and differentiation potential.
2.iPSC culture environment should be isolated from other cells and tested for mycoplasma after two passages.
01Matrigel is used for coating multi-well plates:
1) Place the Gelnest Matrigel (for stem cells) in ice and melt at 4 °C, mixing the Matrigel using a pre-chilled pipette or tip until it is homogeneous.
Note: Gelnest Matrigel is prepared from basement membrane components extracted from mouse tumor tissues, and contains laminin, type IV collagen, heparan sulfate, glycoprotein, etc., and contains a variety of growth factors. These components can provide the support and signals needed for cell adhesion, differentiation, and proliferation, mimicking the properties of basement membranes in a physiological environment, thereby further mimicking cell signaling pathways and interactions in a physiological environment, and improving the success rate and effectiveness of cell culture.
2) On ice, mix Matrigel with DMEM culture medium in a ratio of 1:80 or 1:100, for example, take 1ml Matrigel and add it to 80ml or 100ml DMEM culture medium, and mix it to a homogeneous state using a pre-cooled pipette.
Note: The dilution ratio can be adjusted according to the specific conditions of the experiment.
3) Place the 6-well plate on ice and add the diluted Matrigel to the 6-well plate at a rate of 1 ml per well.
4) Place the 6-well plate in a 37-well incubation overnight.
Note: In general, 1 h incubation is ready to use, but cell culture results are better with overnight incubation.
5) Aspirate the unbound Matrigel before use.
Note: Make sure that the tip of the pipette does not scratch the surface of the coating.
02 Preparation of Y-27632 (Rock inhibitor) solution.
Dissolve Y-27632 in PBS and prepare a 10 mM solution of Y-27632, e.g., take 247 mg Y-27632 dissolved in 1 ml PBS to obtain 1 ml of 10 mM Y-27632 solution.
Note: The working concentration of Y-27632 is 10 M.
03Prepare human embryonic stem cell culture medium containing Y-27632 (e.g., MTERR 1 or TESR-E8).
Take Y-27632 solution and mtesr 1 culture medium and mix it according to the ratio of 1:1000, for example, take 10 L of Y-27632 solution and add 10 ml of mtesr 1 culture medium to 10 ml of mtesr 1 culture medium to obtain 10 ml of mtesr 1 culture medium containing 10 M Y-27632.
04 IPSC Recovery:
1) Place the cryopreserved iPSCs in a 37 water bath and thaw them quickly, transfer the cell solution to a centrifuge tube, rinse the cryovial twice with DMEM culture medium and transfer to a centrifuge tube, merge with the cells in the tube, and centrifuge at room temperature 300 g for 5 min.
2) Discard the supernatant and resuspend the iPSC with 2 ml of Mtesr 1 culture medium containing Y-27632, and then transfer the cell suspension to 1 well of a Matrigel-coated 6-well plate and place it in an incubator.
Note: It is recommended to transfer each strand of recovered cells (approximately 1 million) to 1 well of a 6-well plate for culture to maximize cell viability.
3) The next day, the iPSC was exchanged, and the MTSR 1 culture medium containing Y-27632 was replaced with the MTSR 1 culture medium without Y-27632.
Note: The original cell recovery process can be replaced by the new Nexus cell recovery instrument, which has a short cell recovery time and high cell viability. 
05 IPSC Generation:
Note: iPSC cells differentiate rapidly and die after growth to a monolayer, and are passaged prior to fullness in order to maintain their viability and pluripotency.
1) To remove the supernatant, wash 1 time with 1 ml of PBS and add 1 ml of the appropriate cell digestion solution.
2) Transfer the plate to the incubator and incubate for 3 min. Microscopically, most of the cells are detached, and if the cells are still attached, the bottom of the plate can be gently patted with your hand to cause the cells to fall off.
3) Transfer the digested cells to a centrifuge tube, wash the surface of the culture plate twice with DMEM culture medium and transfer to a centrifuge tube, merge with the cells in the tube, and centrifuge at 300 g for 5 min at room temperature.
4) Discard the supernatant and resuspend with Mtesr 1 medium containing Y-27632, then transfer the cell suspension to a Matrigel-coated 6-well plate and place it in an incubator.
06IPSC cryopreservation:
The Nest Biobank series includes a full range of cell cryopreservation tubes and cryopreservation solutions that are easy to use, safe and sterile.
2) Refer to the relevant steps of the cell cryopreservation solution for cell digestion.
3) Count, discard the supernatant, add an appropriate amount of cryopreservation solution, and aliquot 1ml of cryopreservation solution (about 1 million cells) per tube into cryopreservation tubes.
4) Place the vials in a Nest program cooling box that gradually lowers the temperature and place in the -80 freezer to ensure that the cooling rate is approximately 1 minute. In general, the cooling rate should not be faster than 05 minutes.
5) Place in the -80 refrigerator for about 24 hours and then transfer to the liquid nitrogen vapor phase for storage.
The successful induction of iPS cells has opened up a whole new path for cell** and regenerative medicine research. It is believed that in the near future, iPSC technology can quickly break through the bottleneck, and Nest will continue to develop new products to promote the vigorous development of regenerative medicine and bring new hope to patientsCells**
NEST Matrigel Selection Guide